Blood glucose concentration (blood sugar level) is an important marker for diabetes. Devices for self-monitoring of blood glucose (SMBG) using electrochemical biosensors are widely used by diabetes patients as a device for monitoring their own blood sugar levels. The biosensors used in SMBG devices have conventionally used an enzyme such as glucose oxidase (GOD) that uses glucose as a substrate. However, since GOD has the characteristic of using oxygen as an electron acceptor, SMBG devices using GOD have the potential for preventing the obtaining of accurate measured values due to dissolved oxygen in the measurement sample having an effect on measured values.
On the other hand, various types of glucose dehydrogenases (GDH) are known as enzymes that also use glucose as a substrate but do not use oxygen as an electron acceptor. More specifically, a type of GDH that uses nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme (NAD(P)-GDH) and a type of GDH that uses pyrroloquinoline quinone (PQQ) as coenzyme (PQQ-GDH) have been discovered, and these enzymes are used in the biosensors of SMBG devices. However, NAD(P)-GDH lacks enzyme stability while also having the problem of requiring addition of a coenzyme, while PQQ-GDH has low substrate specificity, causing it to act on sugar compounds other than glucose such as maltose, D-galactose or D-xylose, thereby allowing sugar compounds in measurement samples other than glucose to have an effect on measured values, and resulting in the problem of being unable to obtain accurate measured values.
Recently, PQQ-GDH has been reported to act on maltose contained in transfusion solutions when SMBG devices using PQQ-GDH as a biosensor are used by diabetes patients to measure blood sugar levels, causing measured values to be higher than actual blood sugar levels, and occurrences of disorders such as hypoglycemia caused by treatment based on those values have been reported. In addition, similar occurrences have been determined to also be possible in patients undergoing galactose tolerance tests and xylose absorption tests (see, for example, Non-Patent Document 1). In response to this, when the Pharmaceutical and Food Safety Bureau of the Ministry of Health, Labour and Welfare conducted a cross-reactivity study for the purpose of investigating effects on measured blood sugar levels in the case of having added various sugars to a glucose solution, in the case of adding maltose at 600 mg/dL, D-galactose at 300 mg/dL or D-xylose at 200 mg/dL, measured values obtained with a blood glucose monitoring kit using the PQQ-GDH method were indicated to be nearly 2.5 to 3 times higher than actual glucose concentration. Namely, measured values were determined to be made inaccurate by maltose, D-galactose and D-xylose present in measurement samples, thus resulting in a fervent desire to develop a GDH having high substrate specificity enabling specific measurement of glucose without being affected by sugar compounds causing measurement error in this manner.
With the foregoing in view, attention came to be focused on types of GDH that use coenzymes other than those previously described. For example, GDH derived from Aspergillus oryzae is reported in Non-Patent Documents 2 to 5, while flavin-binding glucose dehydrogenase that uses flavin adenine dinucleotide (FAD) as a coenzyme (to be referred to as FAD-GDH) is disclosed in Patent Documents 1 to 3.
However, although the aforementioned enzymes demonstrate the property of having low reactivity with respect to one or more types of sugar compounds that are not D-glucose, they do not have the property of having sufficiently low reactivity with respect to any of maltose, D-galactose and D-xylose. In contrast, the applicant found that flavin-binding GDHs isolated from the genus Mucor have the superior property of demonstrating sufficiently low reactivity with respect to maltose, D-galactose and D-xylose (see, for example, Patent Document 4). In addition, the use of this GDH was confirmed to enable accurate measurement of D-glucose concentration without being affected by maltose, D-galactose or D-xylose even under conditions in which these sugar compounds are present within a certain concentration range (see, for example, Patent Document 4). This superior substrate specificity is a major characteristic indicating the superiority of these Mucor-derived FAD-GDHs in terms of practical use.
On the other hand, continuing efforts are being made to shorten measurement time by further improving measurement sensitivity, further reduce the scale of measurement systems, and reduce the required size of the measurement sample in order to further improve the convenience of self-monitoring of blood glucose. For example, increasing the amount of glucose measurement enzyme mounted on a glucose sensor has been presumed as a means for improving measurement sensitivity. However, under the predicted conditions of such a usage method in which a large amount of enzyme is present, even in the case of using the previously described Mucor-derived FAD-GDH, reactivity to maltose and D-xylose present at a certain concentration or higher has been confirmed, albeit at a very low level, thus demonstrating that there continues to be room for improvement with respect to efforts to lower reactivity with respect to sugar compounds other than D-glucose.
A method has been disclosed for obtaining modified FAD-GDH in which reactivity to D-xylose has been lowered by introducing an amino acid substitution into Aspergillus-derived FAD-GDH in an attempt to modify existing FAD-GDH for the purpose of improving the substrate specificity of FAD-GDH (see, for example, Patent Documents 5 and 6). However, since Aspergillus-derived FAD-GDH has considerably higher reactivity to D-xylose in comparison with naturally-occurring Mucor-derived FAD-GDH, substrate specificity is still considered to be inadequate even with the previously disclosed modified Aspergillus species-derived FAD-GDH.